Regulatable adeno-associated virus (aav) vector

ABSTRACT

The present invention relates to regulatable adeno-associated virus (AAV) vectors as well as to their use in gene therapy. It further relates to corresponding nucleic acid molecules, host cells, non-human transgenic animals, pharmaceutical compositions and kits.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application claims the benefit of priority from European Patent Application No. 16 166 637.5, filed on Apr. 22, 2016, which is hereby incorporated by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING

A sequence listing containing SEQ ID NOs:1-2 is submitted herewith and is specifically incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to regulatable adeno-associated virus (AAV) vectors as well as to their use in gene therapy. It further relates to corresponding nucleic acid molecules, host cells, non-human transgenic animals, pharmaceutical compositions and kits.

BACKGROUND OF THE INVENTION

Gene therapy in its current configuration is an irreversible process. Typically, a potentially therapeutic transgene is inserted into target cells/tissues by means of gene transfer with a viral vector. From this time on, the transgene is expressed without external control over its expression level and without the option to shut off transgene expression in case of unforeseen side effects or sufficient therapeutic success.

Attempts to overcome this limitation have generated several regulatable gene transfer systems, one of which is the gene switch (GS), whose principles are described in FIG. 1. A constitutive promoter (which may be cell-type specific or ubiquitously active) expresses the GS fusion protein, which consists of a GAL4 DNA binding domain, a truncated progesterone receptor ligand binding domain as drug binding domain, and the p65 transactivation domain from NF-kappaB. Upon binding of the GS fusion protein to the small molecule drug mifepristone (Mfp, a synthetic steroid), it dimerizes and binds to a polynucleotide sequence consisting of several GAL4 DNA binding sites. Consequently, the p65 moiety of the GS fusion protein recruits the basic cellular transcription machinery, allowing expression of the therapeutic factor from a minimal promoter. Withdrawal of Mfp disables binding of the GS fusion protein to its DNA target sequence, and thus ceases transgene expression.

The GS system was described for plasmids, transgenic cells, adenoviral and herpes simplex gene transfer vectors and for transgenic animals during the 1990's (Wang Y. et al., Proc. Natl. Acad. Sci. USA. 1994, 91:8180-8184; Wang Y. et al., Gene Ther. 1997, 4:432-441; Burcin M. M. et al., Proc. Natl. Acad. Sci. USA. 1999, 96:355-360; Oligino T. et al., Gene Ther. 1998, 5:491-496). As adeno-associated viral vectors (AAVs) are especially well-suited gene therapy tools due to their proven safety record in human clinical trials (Bartus R. T. et al., Neurology. 2013, 80:1698-1701; Chtarto A. et al., Br J Clin Pharmacol. 2013, 76:217-232), the inventor has adopted the GS system to AAV vectors. Using the brain as target tissue and Parkinson's disease as a target disease, the inventor demonstrated successful therapeutic treatment of motor symptoms in a rat model of Parkinson's disease (Tereshchenko J. et al., Neurobiol Dis. 2014, 65:35-42; Maddalena A. et al., Mol Ther Nucleic Acids. 2013, 2:e106). In this approach, the inventor was able to show that short-term induced expression of the neurotrophic factor GDNF (glial cell line-derived neurotrophic factor, Kordower J. H. et al., Mov Disord. 2013, 28:96-109) resulted in long-term recovery from motor impairments in this model.

A functional GS system usually requires gene transfer of two expression cassettes into target cells: one cassette expressing the GS fusion protein and a second cassette expressing the therapeutic factor from the regulated minimal promoter. Accordingly, the above-mentioned studies were conducted with a two-vector system, in which the GS expression cassette was contained in one virus, while the regulated GDNF expression cassette was contained in a second virus. This configuration was necessary to prevent leaky expression of GDNF in the non-Mfp-induced state (Maddalena A. et al., Mol Ther Nucleic Acids. 2013, 2:e106). While such a two-vector system offers flexibility in terms of adjusting the ratio of GS expression cassette versus the GDNF expression cassette, it appears unlikely that such a double vector formulation could be approved by authorities for human gene therapy.

A one-vector system described in Maddalena A. et al. (Mol Ther Nucleic Acids. 2013, 2:e106) exhibits leaky expression of GDNF in the non-Mfp-induced state. Other regulated vector systems, e.g., as described in Liu Y. et al. (Molecular Therapy. 2008, 16(3):474-480) and Naidoo J. et al. (Neurology Research Int. 2012, 267, No. 5202), are based on the use of active transcriptional silencers, such as the Tet repressor, and/or are based on viruses, which will hamper or prevent the clinical use of these systems.

Thus, there is a need in the art for a regulated AAV vector-based expression system with at least the following features:

-   -   all elements of the system (e.g., GS system) for regulated         expression of a therapeutic molecule (e.g., a neurotrophic         factor, such as GDNF) should be contained in one vector genome;     -   after injection of this vector into the patient (e.g., into a         specific tissue/organ, such as the brain), it should not express         the therapeutic molecule in the absence of the activator         molecule (e.g., Mfp);     -   after application of the activator molecule, levels of the         therapeutic molecule should increase substantially to levels of         therapeutic value; and     -   after withdrawal of the activator molecule levels of the         therapeutic molecule should decline to background levels again.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to an adeno-associated virus (AAV) vector comprising

(i) a first expression cassette directing the expression of a regulator protein under the control of a first promoter, wherein the regulator protein is activated in the presence of an activator molecule, and (ii) a second expression cassette directing the expression of a molecule of interest, wherein the second expression cassette comprises a promoter region, and the expression of the molecule of interest is induced by binding of the activated regulator protein to the promoter region, wherein the first expression cassette and the second expression cassette are arranged in a tail-to-head configuration.

In one embodiment, the first promoter has one or more of the following features:

(i) it is a constitutive promoter; (ii) it is selected from the group consisting of cell-specific promoters, tissue-specific promoters and organ-specific promoters; (iii) it is selected from the group consisting of human synapsin 1 gene (hSYN1) promoter, tubulin alpha 1 (Ta1) promoter, glial fibrillary acidic protein (GFAP) promoter, cytomegalovirus (CMV) promoter, human beta-actin-CMV hybrid promoter and functional fragments or variants of any of the foregoing.

In one embodiment, the regulator protein is a gene switch fusion protein comprising a GAL4 DNA binding domain, a truncated progesterone receptor ligand binding domain and a p65 transactivation domain from NF-kappaB.

In one embodiment, the activator molecule is mifepristone (Mfp).

In one embodiment, the first expression cassette comprises, in 5′ to 3′ direction, the first promoter, a coding sequence for the regulator protein and a first polyadenylation signal sequence, wherein, optionally, the first expression cassette further comprises a synthetic intron arranged between the coding sequence for the regulator protein and the first polyadenylation signal sequence.

In one embodiment, the promoter region comprises one or more binding sites for the activated regulator protein, a second promoter and, optionally, a synthetic intron.

In one embodiment, the one or more binding sites for the activated regulator protein are GAL4 binding sites.

In one embodiment, the second promoter is a minimal promoter which is induced by the binding of the activated regulator protein to the one or more binding sites for the activated regulator protein.

In one embodiment, the second promoter is a minimal promoter comprising a TATA sequence and/or an mRNA initiation sequence.

In one embodiment, the molecule of interest is a therapeutically active peptide or protein or a therapeutically active oligo- or polynucleotide.

In one embodiment, the molecule of interest is a neurotrophic factor.

In one embodiment, the second expression cassette comprises, in 5′ to 3′ direction, the promoter region, a coding sequence for the molecule of interest and a second polyadenylation signal sequence.

In one embodiment, the first expression cassette and the second expression cassette are separated by a nucleotide sequence comprising an insulator element.

In one embodiment, the insulator element is a transcription blocker comprising a transcription pause site and a polyadenylation signal sequence.

In one embodiment, the AAV vector comprises the nucleotide sequence represented by SEQ ID NO: 1 or a functional variant thereof, wherein the functional variant has a nucleotide sequence which is at least 80% or at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 1.

In one embodiment, in the absence of the activator molecule, the molecule of interest is not expressed in a host or is expressed in a host at a level which is at most 10-fold or at most 5-fold or at most 4-fold or at most 3-fold or at most 2-fold increased as compared to the normal expression level of the molecule of interest in the host, wherein the host is a cell, tissue or organ.

In another aspect, the present invention relates to an adeno-associated virus (AAV) vector construct comprising

(i) a first expression cassette directing the expression of a regulator protein under the control of a first promoter, wherein the regulator protein is activated in the presence of an activator molecule, and (ii) a second expression cassette comprising a multiple cloning site allowing the insertion of a coding sequence for a molecule of interest, wherein the second expression cassette comprises a promoter region, and the expression of the molecule of interest is induced by binding of the activated regulator protein to the promoter region, wherein the first expression cassette and the second expression cassette are arranged in a tail-to-head configuration.

In one embodiment, the AAV vector construct comprises the nucleotide sequence represented by SEQ ID NO: 2 or a functional variant thereof, wherein the functional variant has a nucleotide sequence which is at least 80% or at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 2.

In another aspect, the present invention relates to the AAV vector as defined above, wherein the molecule of interest is a therapeutically active peptide or protein or a therapeutically active oligo- or polynucleotide, for use as a medicament.

In another aspect, the present invention relates to the AAV vector as defined above, wherein the molecule of interest is a neurotrophic factor, for use in treating, ameliorating or preventing a disease or disorder selected from the group consisting of Parkinson's disease, Huntington's disease, spinal cord lesion and an amyloid-related disorder, wherein, preferably, the amyloid-related disorder is selected from the group consisting of Alzheimer's disease (e.g., sporadic Alzheimer's disease or familial Alzheimer's disease), cerebral amyloid angiopathy, dementia, motor neuropathy, Down's syndrome, Creutzfeld Jacob disease, transmissible spongiform encephalopathies, hereditary cerebral hemorrhage with amyloidosis Dutch type, HIV-related dementia, fronto-temporal dementia, Lewy body disease, mixed dementias, head trauma, familial Danish Dementia, familial British Dementia, inclusion body myositis (IBM), neuronal disorder related to aging, and chronic pain.

In another aspect, the present invention relates to the use of the AAV vector as defined above for the preparation of a medicament for treating, ameliorating or preventing a disease or disorder in a subject, wherein the molecule of interest is a therapeutically active peptide or protein or therapeutically active oligo- or polynucleotide.

In another aspect, the present invention relates to a method for treating, ameliorating or preventing a disease or disorder in a subject, comprising

(a) introducing into the subject the AAV vector as defined above; and (b) administering to the subject the activator molecule to induce expression of the molecule of interest, wherein the molecule of interest is a therapeutically active peptide or protein or therapeutically active oligo- or polynucleotide.

In some embodiments of said use or said method, the molecule of interest is a neurotrophic factor and the disease or disorder is selected from the group consisting of Parkinson's disease, Huntington's disease, spinal cord lesion and an amyloid-related disorder, wherein, preferably, the amyloid-related disorder is selected from the group consisting of Alzheimer's disease (e.g., sporadic Alzheimer's disease or familial Alzheimer's disease), cerebral amyloid angiopathy, dementia, motor neuropathy, Down's syndrome, Creutzfeld Jacob disease, transmissible spongiform encephalopathies, hereditary cerebral hemorrhage with amyloidosis Dutch type, HIV-related dementia, fronto-temporal dementia, Lewy body disease, mixed dementias, head trauma, familial Danish Dementia, familial British Dementia, inclusion body myositis (IBM), neuronal disorder related to aging, and chronic pain.

In another aspect, the present invention relates to a host cell comprising the AAV vector as defined above.

In another aspect, the present invention relates to a non-human transgenic animal comprising the AAV vector as defined above.

In another aspect, the present invention relates to a pharmaceutical composition comprising the AAV vector as defined above or the host cell as defined above.

In yet another aspect, the present invention relates to a kit comprising the AAV vector as defined above or the AAV vector construct as defined above or the host cell as defined above or the pharmaceutical composition as defined above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the components of the gene switch (GS) system.

FIG. 2 shows different one-vector GS-GDNF genomes tested by the inventor. SV40=simian virus 40 polyadenylation site; Intron=polynucleotide containing splice donor and acceptor sites; GS=cDNA of the gene switch fusion protein; hSYN1=fragment of human synapsin 1 gene promoter; uasTATA=regulated promoter consisting of six GAL4 binding sites and a minimal TATA promoter plus splice donor and acceptor sites; GDNF=cDNA of GDNF; BGH=bovine growth hormone polyadenylation site; TB=polynucleotide containing synthetic transcription pause and polyadenylation site.

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms (IUPAC Recommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995). The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 3^(rd) Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2000).

In the following, certain elements of the present invention will be described. These elements may be listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments, which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps although in some embodiments such other member, integer or step or group of members, integers or steps may be excluded, i.e. the subject-matter consists in the inclusion of a stated member, integer or step or group of members, integers or steps. The terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The present invention provides an adeno-associated virus (AAV) vector comprising

(i) a first expression cassette directing the expression of a regulator protein under the control of a first promoter, wherein the regulator protein is activated in the presence of an activator molecule, and (ii) a second expression cassette directing the expression of a molecule of interest, wherein the second expression cassette comprises a promoter region, and the expression of the molecule of interest is induced by binding of the activated regulator protein to the promoter region, wherein the first expression cassette and the second expression cassette are arranged in a tail-to-head configuration.

As used herein, the term “adeno-associated virus (AAV) vector” means an AAV viral particle containing an AAV vector genome (which, in turn, comprises the first and second expression cassettes referred to herein). It is meant to include AAV vectors of all serotypes, preferably AAV-1 through AAV-9, more preferably AAV-1, AAV-2, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and combinations thereof. AAV vectors resulting from the combination of different serotypes may be referred to as hybrid AAV vectors. In one embodiment, the AAV vector is selected from the group consisting of AAV-1, AAV-2, AAV-4, AAV-5 and AAV-6, and combinations thereof. In one embodiment, the AAV vector is an AAV-5 vector. In one embodiment, the AAV vector is an AAV-5 vector comprising AAV-2 inverted terminal repeats (ITRs). Also included in the present invention are AAV vectors comprising variants of the naturally occurring viral proteins, e.g., one or more capsid proteins. In one embodiment, the one or more variant capsid proteins comprise the substitution of one or more amino acid residues, thereby modifying, preferably improving, the vector transduction properties. Suitable variants of AAV viral proteins are known to the skilled person.

The term “expression cassette”, as used herein, refers to a nucleotide sequence which directs the cell's machinery (or any other transcription active system, such as an in vitro transcription/translation system) to express/make a particular functional product. Generally, an expression cassette comprises a promoter sequence, an open reading frame (also referred to as coding region) coding, e.g., for a peptide or protein, and a 3′ untranslated region (3′ UTR), which preferably contains a polyadenylation signal sequence. An expression cassette according to the present invention may further comprise a 5′ untranslated region (5′ UTR), which is located 3′ of the promoter (region) and 5′ of the coding region.

The term “tail-to-head configuration”, as used herein, means that, in the AAV vector, (i) the 3′ end of the first expression cassette is adjacent to the 5′ end of the second expression cassette or (ii) the 3′ end of the second expression cassette is adjacent to the 5′ end of the first expression cassette. In other words, the first expression cassette and the second expression cassette are oriented in the same transcriptional direction. In a preferred embodiment, the 3′ end of the first expression cassette is adjacent to the 5′ end of the second expression cassette.

In accordance with the present invention, “adjacent” can be “directly adjacent” or “indirectly adjacent”. The term “indirectly adjacent”, as used herein, refers to the situation where the first expression cassette and the second expression cassette are separated by a nucleotide sequence. In one embodiment, the nucleotide sequence consists of less than 1500 nucleotides, less than 1000 nucleotides, less than 500 nucleotides, less than 250 nucleotides, less than 200 nucleotides, or less than 150 nucleotides. In one embodiment, the nucleotide sequence comprises, essentially consists of or consists of an insulator element.

The term “insulator element”, as used herein, refers to a nucleotide sequence, preferably a synthetic nucleotide sequence, reducing or preventing expression of the molecule of interest in the absence of the activator molecule. Such nucleotide sequences are known to a person skilled in the art. In one embodiment, the insulator element is a transcription blocker (TB) comprising, essentially consisting of or consisting of a transcription pause site and a polyadenylation signal sequence (also referred to herein as a polyadenylation site).

In one embodiment, the first promoter is a constitutive promoter. The term “constitutive promoter”, as used herein, refers to an unregulated promoter that allows for continual transcription of its associated gene.

In one embodiment, the first promoter is selected from the group consisting of human synapsin 1 gene (hSYN1) promoter, tubulin alpha 1 (Ta1) promoter, glial fibrillary acidic protein (GFAP) promoter, cytomegalovirus (CMV) promoter, human beta-actin-CMV hybrid promoter and functional fragments or variants of any of the foregoing.

In one embodiment, the first promoter allows for ubiquitous expression of the regulator protein and is preferably selected from the group consisting of cytomegalovirus (CMV) promoter, human beta-actin-CMV hybrid promoter and functional fragments or variants of any of the foregoing.

In one embodiment, the first promoter is selected from the group consisting of cell-specific promoters, tissue-specific promoters and organ-specific promoters. In one embodiment, the promoter is a neuron-specific promoter or an astrocyte-specific promoter. In one embodiment, the neuron-specific promoter is selected from the group consisting of human synapsin 1 gene (hSYN1) promoter, tubulin alpha 1 (Ta1) promoter and functional fragments or variants of any of the foregoing. In one embodiment, the neuron-specific promoter is human synapsin 1 gene (hSYN1) promoter or a functional fragment or variant thereof. In one embodiment, the astrocyte-specific promoter is glial fibrillary acidic protein (GFAP) promoter or a functional fragment or variant thereof.

The term “regulator protein”, as used herein, refers to a protein (e.g., a fusion protein), which, upon interaction with an activator molecule (in particular, upon binding of the activator molecule to the regulator protein), undergoes a conformational change to an activated state and binds to the promoter region of the second expression cassette, thereby inducing the expression of the molecule of interest. According to the present invention, the regulator protein does not bind to the promoter region of the second expression cassette in its inactive state. In one embodiment, the regulator protein is a fusion protein comprising a (truncated) progesterone receptor ligand binding domain. In one embodiment, the fusion protein is a gene switch fusion protein. Suitable gene switch fusion proteins that can be used in accordance with the present invention are described, for example, in WO 2002/24899 A2, WO 2009/045370 A2, Wang Y. et al., Proc. Natl. Acad. Sci. USA. 1994, 91:8180-8184, and Wang Y. et al., Gene Ther. 1997, 4:432-441. In one embodiment, the regulator protein is a gene switch fusion protein comprising, essentially consisting of or consisting of a GAL4 DNA binding domain (e.g., amino acids 1 to 93 of S. cerevisiae GAL4), a truncated progesterone receptor ligand binding domain (e.g., amino acids 640 to 914 of human progesterone receptor) and a p65 transactivation domain from NF-kappaB (e.g., amino acids 283 to 551 of the human p65 subunit of NF-kappaB).

The term “activator molecule”, as used herein, refers to a molecule (e.g., a small molecule compound) that activates the regulator protein in vivo and is, preferably, pharmaceutically acceptable. Preferably, the activator molecule is a ligand of the regulator protein, wherein activation of the regulator protein may, for example, comprise the dimerization of the regulator protein. Suitable activator molecules that can be used in accordance with the present invention are described, for example, in WO 2002/24899 A2, WO 2009/045370 A2, Wang Y. et al., Proc. Natl. Acad. Sci. USA. 1994, 91:8180-8184, and Wang Y. et al., Gene Ther. 1997, 4:432-441. In one embodiment, the activator molecule is an anti-progestin. In one embodiment, the activator molecule is mifepristone (Mfp; RU486).

A commercially available gene switch system that can be used in accordance with the present invention is the GeneSwitch™ System (ThermoFisher Scientific).

The AAV vector of the present invention may comprise one or more introns. The term “intron” as used herein refers to a sequence encoded in a DNA sequence that is transcribed into an RNA molecule by RNA polymerase but is removed by splicing to form the mature messenger RNA. A “synthetic intron” refers to a sequence that is not initially replicated from a naturally occurring intron sequence and generally will not have a naturally occurring sequence, but will be removed from an RNA transcript during normal post-transcriptional processing. Preferably, the synthetic intron includes consensus sequences for the 5′ splice site, 3′ splice site, and, optionally, the branch point. Such synthetic intron, when introduced into the AAV vector of the present invention, may direct the splicing of RNA transcripts in a highly efficient and accurate manner, thereby minimizing cryptic splicing and maximizing production of the desired gene product, e.g., the regulator protein and/or the molecule of interest. In one embodiment, the synthetic intron is an IVS8 synthetic intron.

In one embodiment, the AAV vector of the present invention does not comprise a(n) (active) transcriptional silencer, such as a Tet repressor.

In one embodiment, the first expression cassette comprises or essentially consists of, in 5′ to 3′ direction, the first promoter, a coding sequence for the regulator protein and a first polyadenylation signal sequence. Optionally, the first expression cassette further comprises a synthetic intron arranged between the coding sequence for the regulator protein and the first polyadenylation signal sequence and/or a synthetic intron located in the 5′ UTR, if present.

In one embodiment, the promoter region in the second expression cassette comprises, essentially consists of or consists of one or more binding sites for the activated regulator protein, a second promoter and, optionally, a synthetic intron.

In one embodiment, the one or more binding sites for the activated regulator protein are GAL4 binding sites, also referred to herein as GAL4 upstream activating sequences (UAS). In one embodiment, the promoter region comprises six GAL4 binding sites.

In one embodiment, the second promoter is a minimal promoter which is induced by the binding of the activated regulator protein to the one or more binding sites for the activated regulator protein. The term “minimal promoter”, as used herein, refers to minimal portion of a promoter required to properly initiate transcription. In one embodiment, the second promoter is a minimal promoter comprising, essentially consisting of or consisting of a TATA sequence and/or an mRNA initiation sequence. In one embodiment, the second promoter is a minimal promoter comprising the adenovirus E1b TATA sequence.

In one embodiment, the second expression cassette comprises, essentially consists of or consists of, in 5′ to 3′ direction, the promoter region, a coding sequence for the molecule of interest and a second polyadenylation signal sequence. The second expression cassette may also comprise one or more synthetic introns, e.g., in the promoter region and/or arranged between the coding sequence for the molecule of interest and the second polyadenylation signal sequence.

According to the present invention, the first polyadenylation signal sequence and the second polyadenylation signal sequence may be the same or different. In one embodiment, the first polyadenylation signal sequence and the second polyadenylation signal sequence are independently selected from the group consisting of a simian virus 40 (SV40) polyadenylation signal sequence, a bovine growth hormone (bGH) polyadenylation signal sequence and a human growth hormone (hGH) polyadenylation signal sequence. In one embodiment, the first polyadenylation signal sequence is a simian virus 40 (SV40) polyadenylation signal sequence, and the second polyadenylation signal sequence is a bovine growth hormone (bGH) polyadenylation signal sequence.

In the context of the present invention, the term “DNA” relates to a molecule, which comprises deoxyribonucleotide residues and preferably is entirely or substantially composed of deoxyribonucleotide residues. “Deoxyribonucleotide” relates to a nucleotide, which lacks a hydroxyl group at the 2′-position of a β-D-ribofuranosyl group. The term “DNA” comprises isolated DNA such as partially or completely purified DNA, essentially pure DNA, synthetic DNA, and recombinantly generated DNA and includes modified DNA, which differs from naturally occurring DNA by addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a DNA or internally, for example at one or more nucleotides of the DNA. Nucleotides in DNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides. These altered DNAs can be referred to as analogs or analogs of naturally occurring DNA.

The term “molecule of interest”, as used herein, refers to a macromolecule, such as but not limited to RNA, a peptide and a polypeptide or protein.

In one embodiment, the molecule of interest is a therapeutically active peptide or protein or a therapeutically active oligo- or polynucleotide. The term “peptide”, as used herein, generally relates to substances which include at least 2, at least 3, at least 4, at least 6, at least 8, at least 10, at least 12 or at least 14 and preferably up to 8, 10, 12, 14, 16, 18, 20, 25, 30, 50, or 100 consecutive amino acids which are connected together by peptide bonds. The terms “polypeptide” and “protein”, as used herein, relate to large peptides, preferably peptides having more than 100 amino acids, but the terms “peptide”, “polypeptide” and “protein” may be used interchangeably herein. The term “oligonucleotide”, as used herein, refers to short DNA or RNA molecules, preferably with 30 or less nucleotide residues. The term “polynucleotide”, as used herein, refers to long DNA or RNA molecules, preferably with more than 30 nucleotide residues. In one embodiment, the oligo- or polynucleotide is an RNA oligo- or polynucleotide.

In the context of the present invention, the term “RNA” relates to a molecule, which comprises ribonucleotide residues and preferably is entirely or substantially composed of ribonucleotide residues. “Ribonucleotide” relates to a nucleotide with a hydroxyl group at the 2′-position of a β-D-ribofuranosyl group. The RNA may, for example, be a ribozyme, an antisense RNA or an miRNA.

The term “therapeutically active”, as used herein, refers to a molecule of interest which has a therapeutic/pharmacologic effect when administered appropriately to a subject suffering from a disease or disorder. Such therapeutic/pharmacologic effect is one that is expected to be related to a beneficial effect on the course or a symptom of the disease or disorder.

In one embodiment, the molecule of interest is a neurotrophic factor. The term “neurotrophic factor”, as used herein, refers to proteins that are involved in or responsible for the growth and survival of developing neurons and the maintenance of mature neurons. In one embodiment, the neurotrophic factor is selected from the group consisting of glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), nerve growth factor (NGF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN). GDNF, NRTN, ARTN and PSPN are members of the GDNF family of ligands (GFL), which may be preferred neurotrophic factors in accordance with the present invention. In one embodiment, the neurotrophic factor is GDNF.

The molecule of interest may, in accordance with the present invention, also be a peptide or protein or a oligo- or polynucleotide, which is not therapeutically active. Such molecule of interest may, for example, be a fluorescent protein, such as (E)GFP, RFP, YFP and derivatives thereof, or a luminescent protein, such as luciferase and derivatives thereof.

In one embodiment, the AAV vector comprises the nucleotide sequence represented by SEQ ID NO: 1 (or a nucleotide sequence complementary to SEQ ID NO: 1) or a functional variant thereof, wherein the functional variant has a nucleotide sequence which is at least 80% or at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 1 (or to the nucleotide sequence complementary to SEQ ID NO: 1).

In one embodiment, the AAV vector further comprises inverted terminal repeats (ITRs), e.g., AAV-2 ITRs, flanking the first and second expression cassettes (or the nucleotide sequence defined above, if applicable).

The present invention also provides an AAV vector as defined herein, wherein the molecule of interest is a therapeutically active peptide or protein or a therapeutically active oligo- or polynucleotide, for use as a medicament. The term “medicament”, as used herein, refers to a substance/composition used in therapy, i.e., in treating, ameliorating or preventing a disease or disorder. According to the invention, the terms “disease” or “disorder” refer to any pathological state. In one embodiment, the disease or disorder is a neurological or neurodegenerative disease.

The present invention also provides an AAV vector as defined herein, wherein the molecule of interest is a neurotrophic factor, for use in treating, ameliorating or preventing a disease or disorder selected from the group consisting of Parkinson's disease, Huntington's disease, spinal cord lesion and an amyloid-related disorder, wherein, preferably, the amyloid-related disorder is selected from the group consisting of Alzheimer's disease (e.g., sporadic Alzheimer's disease or familial Alzheimer's disease), cerebral amyloid angiopathy, dementia, motor neuropathy, Down's syndrome, Creutzfeld Jacob disease, transmissible spongiform encephalopathies, hereditary cerebral hemorrhage with amyloidosis Dutch type, HIV-related dementia, fronto-temporal dementia, Lewy body disease, mixed dementias, head trauma, familial Danish Dementia, familial British Dementia, inclusion body myositis (IBM), neuronal disorder related to aging, and chronic pain.

In one embodiment, the neurotrophic factor is selected from the group consisting of GDNF, BDNF, NT-3, NGF, NRTN, ARTN and PSPN, and the AAV vector is for use in treating, ameliorating or preventing a disease or disorder selected from the group consisting of Parkinson's disease, Huntington's disease and an amyloid-related disorder, wherein, preferably, the amyloid-related disorder is selected from the group consisting of Alzheimer's disease (e.g., sporadic Alzheimer's disease or familial Alzheimer's disease), cerebral amyloid angiopathy, dementia, motor neuropathy, Down's syndrome, Creutzfeld Jacob disease, transmissible spongiform encephalopathies, hereditary cerebral hemorrhage with amyloidosis Dutch type, HIV-related dementia, fronto-temporal dementia, Lewy body disease, mixed dementias, head trauma, familial Danish Dementia, familial British Dementia, inclusion body myositis (IBM), neuronal disorder related to aging, and chronic pain. In one embodiment, the disease or disorder is Parkinson's disease.

In one embodiment, the neurotrophic factor is BDNF, and the AAV vector is for use in treating, ameliorating or preventing a spinal cord lesion.

The present invention further provides the use of the AAV vector as defined herein for the preparation of a medicament for treating, ameliorating or preventing a disease or disorder in a subject, wherein the molecule of interest is a therapeutically active peptide or protein or therapeutically active oligo- or polynucleotide.

The present invention further provides a method for treating, ameliorating or preventing a disease or disorder in a subject, comprising

(a) introducing into the subject the AAV vector as defined herein; and (b) administering to the subject the activator molecule to induce expression of the molecule of interest, wherein the molecule of interest is a therapeutically active peptide or protein or therapeutically active oligo- or polynucleotide.

According to the present invention, step (a) may be performed in vivo (by introducing the AAV vector directly into cells of the subject) or at least partially ex vivo (by transferring the AAV vector into isolated cells of the subject or non-autologous cells and introducing the modified cells into the subject or into a different subject). Said introducing may be performed by any suitable method, either systemically (e.g., orally, intravenously, sublingually, transdermally) or locally (e.g., intraperitoneally, intrathecally, intraventricularly or by direct injection into the target tissue or organ). In one embodiment, the AAV vector is introduced into the subject by injection, e.g., by direct injection into the target tissue or organ (e.g., the brain).

The activator molecule may be administered by any suitable method, either systemically (e.g., orally, intravenously, sublingually, transdermally) or locally (e.g., intraperitoneally, intrathecally, intraventricularly or by direct injection into the tissue or organ where the AAV vector was introduced). Administration of the activator molecule can occur once or several times, continuously or intermittently.

The optimal time interval between step (a) and step (b) can be determined for each type of cell/tissue/organ and disease or disorder using only routine techniques.

The term “subject”, as used herein, relates to any organism such as a vertebrate, particularly any mammal, including both a human and another mammal, e.g., an animal such as a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a non-human primate (e.g., a monkey). The rodent may be a mouse, rat, hamster, guinea pig, or chinchilla. Preferably, the subject is a human. In one embodiment, a subject is a subject with or suspected of having a disease or disorder, in particular a disease or disorder as disclosed herein, also designated “patient” herein.

In some embodiments of said use or said method, the molecule of interest is a neurotrophic factor and the disease or disorder is selected from the group consisting of Parkinson's disease, Huntington's disease, spinal cord lesion and an amyloid-related disorder, wherein, preferably, the amyloid-related disorder is selected from the group consisting of Alzheimer's disease (e.g., sporadic Alzheimer's disease or familial Alzheimer's disease), cerebral amyloid angiopathy, dementia, motor neuropathy, Down's syndrome, Creutzfeld Jacob disease, transmissible spongiform encephalopathies, hereditary cerebral hemorrhage with amyloidosis Dutch type, HIV-related dementia, fronto-temporal dementia, Lewy body disease, mixed dementias, head trauma, familial Danish Dementia, familial British Dementia, inclusion body myositis (IBM), neuronal disorder related to aging, and chronic pain.

The present invention also provides nucleic acid molecules, in particular DNA molecules, comprised in the AAV vector of the invention. In one embodiment, said nucleic acid molecule is an AAV vector genome comprising the first expression cassette and the second expression cassette as defined herein, wherein the first expression cassette and the second expression cassette are arranged in a tail-to-head configuration. Such nucleic acid molecules are, e.g., provided in the form of an adeno-associated virus (AAV) vector construct, which, preferably, allows the production of the AAV vector of the invention by methods known in the art (e.g., as described in Tereshchenko J. et al., Neurobiol Dis. 2014, 65:35-42; Maddalena A. et al., Mol Ther Nucleic Acids. 2013, 2:e106; and Drinkut A. et al., Mol Ther. 2012, 20:534-543). Such AAV vector construct may, for example, be in the form of a plasmid (referred to as AAV vector plasmid) or in the form of a linear (expression) construct. The term “AAV vector plasmid”, as used herein, is meant to refer to a double stranded circular nucleic acid molecule that contains at least a functional portion of an AAV nucleic acid molecule.

The present invention further provides an adeno-associated virus (AAV) vector construct comprising

(i) a first expression cassette directing the expression of a regulator protein under the control of a first promoter, wherein the regulator protein is activated in the presence of an activator molecule, and (ii) a second expression cassette comprising a multiple cloning site allowing the insertion of a coding sequence for a molecule of interest, wherein the second expression cassette comprises a promoter region, and the expression of the molecule of interest is induced by binding of the activated regulator protein to the promoter region, wherein the first expression cassette and the second expression cassette are arranged in a tail-to-head configuration.

Such AAV may be referred to as an empty AAV vector construct, i.e., without a coding sequence for a molecule of interest. Preferably, all elements of this empty AAV vector construct are as defined herein in connection with the AAV vector.

In one embodiment, the AAV vector construct comprises the nucleotide sequence represented by SEQ ID NO: 2 (or a nucleotide sequence complementary to SEQ ID NO: 2) or a functional variant thereof, wherein the functional variant has a nucleotide sequence which is at least 80% or at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 2 (or to the nucleotide sequence complementary to SEQ ID NO: 2).

In one embodiment, the AAV vector construct further comprises inverted terminal repeats (ITRs), e.g., AAV-2 ITRs, flanking the first and second expression cassettes (or the nucleotide sequence defined above, if applicable).

In one embodiment, the AAV vector construct comprises a coding sequence for a molecule of interest, which is inserted into said multiple cloning site.

In one embodiment, the AAV vector construct does not comprise a(n) (active) transcriptional silencer, such as a Tet repressor.

Preferably, the AAV vectors of the present invention ensure that, in the absence of the activator molecule, the molecule of interest is not expressed in a host or is expressed in a host at a level which is at most 10-fold or at most 5-fold or at most 4-fold or at most 3-fold or at most 2-fold increased as compared to the normal expression level of the molecule of interest in the host, wherein the host is a cell, tissue or organ.

The terms “part” or “fragment” or “portion” are used interchangeably herein and refer to a continuous element. For example, a part of a structure, such as an amino acid sequence or nucleotide sequence, refers to a continuous element of said structure. For example, a part or fragment of a nucleotide sequence preferably comprises a sequence of at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, at least 100, at least 150, at least 160, at least 170, at least 180, at least 190 or at least 200 consecutive nucleotides of the nucleotide sequence.

For the purposes of the present invention, “variants” of an amino acid sequence or nucleotide sequence comprise amino acid/nucleotide insertion variants, amino acid/nucleotide addition variants, amino acid/nucleotide deletion variants and/or amino acid/nucleotide substitution variants. Amino acid/nucleotide insertion variants comprise insertions of single or two or more amino acids/nucleotides in a particular amino acid sequence or nucleotide sequence. In the case of amino acid/nucleotide sequence variants having an insertion, one or more amino acid/nucleotide residues are inserted into a particular site in an amino acid sequence or nucleotide sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid/nucleotide addition variants comprise N- and/or C-terminal fusions of one or more amino acids/nucleotides, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids/nucleotides. Amino acid/nucleotide deletion variants are characterized by the removal of one or more amino acids/nucleotides from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids/nucleotide. The deletions may be in any position of the amino acid sequence or nucleotide sequence, for example at the N- and/or C-terminus. Amino acid/nucleotide deletion variants that comprise the deletion at the N-terminal and/or C-terminal end of the amino acid sequence or nucleotide sequence are also called N-terminal and/or C-terminal truncation variants. Amino acid/nucleotide substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. In one embodiment, the amino acid/nucleotide substitution variant comprises the substitution of up to 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acids/nucleotides.

The term “variant”, as used herein in connection with a promoter, may also refer to mutants, species variants and homologues of said promoter, including those, which occur naturally.

Alternatively or additionally, a “variant” as used herein, can be characterized by a certain degree of sequence identity to the parent amino acid sequence or nucleotide sequence from which it is derived. More precisely, an amino acid sequence variant in the context of the present invention may exhibit at least 80% sequence identity to its parent amino acid sequence. A nucleotide sequence variant in the context of the present invention may exhibit at least 80% sequence identity to its parent nucleotide sequence. The term “at least 80% identical to”, as used herein, refers to a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective parent/reference amino acid sequence or to the respective parent/reference nucleotide sequence. Preferably, the amino acid sequence in question and the parent/reference amino acid sequence exhibit the indicated sequence identity over the entire length of the parent/reference amino acid sequence. Preferably, the nucleotide sequence in question and the parent/reference nucleotide sequence exhibit the indicated sequence identity over the entire length of the parent/reference nucleotide sequence.

The similarity of nucleotide and amino acid sequences, i.e., the percentage of sequence identity, can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson J. D. et al. Nucleic Acids Res. 1994, 22:4673-80) available e.g. on http://www.ebi.ac.uk/Tools/clustalw/ or on http://www.ebi.ac.uk/Tools/clustalw2/index.html or on http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html. Preferred parameters used are the default parameters as they are set on http://www.ebi.ac.uk/Tools/clustalw/ or http://www.ebi.ac.uk/Tools/clustalw2/index.html. The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. J. Mol. Biol. 1990, 215:403-410. BLAST polynucleotide searches are performed with the BLASTN program, score=100, word length=12, to obtain polynucleotide sequences that are homologous to those nucleic acids which encode F, N, or M2-1. BLAST protein searches are performed with the BLASTP program, score=50, word length=3, to obtain amino acid sequences homologous to the F polypeptide, N polypeptide, or M2-1 polypeptide. To obtain gapped alignments for comparative purposes, Gapped BLAST is utilized as described in Altschul et al. Nucleic Acids Res. 1997, 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:I54-I62) or Markov random fields.

The terms “functional fragment” and “functional variant”, as used herein in connection with a promoter, refers to a fragment or variant of a promoter which is functional in the sense that it has the same or essentially the same activity (in particular properly initiating transcription and/or cell-/tissue-/organ-specificity, if applicable) as said promoter. The term “functional variant”, as used herein in connection with the nucleotide sequence comprised in an AAV vector of the present invention (e.g., the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2), refers to a variant of said nucleotide sequence which is functional in the sense that it allows the regulatable expression of a molecule of interest to the same or essentially the same degree as the AAV vector comprising said nucleotide sequence (e.g., the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2), wherein, preferably, in the absence of the activator molecule, the molecule of interest is not expressed in a host or is expressed in a host at a level which is at most 10-fold or at most 5-fold or at most 4-fold or at most 3-fold or at most 2-fold increased as compared to the normal expression level of the molecule of interest in the host, wherein the host is a cell, tissue or organ.

The present invention also provides a host cell comprising the AAV vector of the present invention or a nucleic acid molecule of the present invention, e.g., an AAV vector construct as defined herein. Such host cell may either be a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell (e.g., a fungal, plant or animal cell). Preferably, the host cell is an isolated host cell. In one embodiment, the host cell is a producer cell (or producer cell line) allowing the production of the AAV vector of the present invention, e.g., based on an AAV vector construct as defined herein and with co-transfection of suitable helper constructs, e.g., helper plasmids (see, for example, US 2004/0235174 A1).

Suitable producer cells are known to a person skilled in the art and include, for example, HEK293 cells.

The present invention further provides a non-human transgenic animal comprising the AAV vector of the present invention. The term “non-human transgenic animal”, as used herein, relates, in particular, to non-human mammals, e.g., a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a non-human primate (e.g., a monkey). The rodent may be a mouse, rat, hamster, guinea pig, or chinchilla.

The present invention further provides a pharmaceutical composition comprising the AAV vector or the host cell of the present invention. A pharmaceutical composition in accordance with the present invention may further comprise one or more carriers and/or excipients, all of which are preferably pharmaceutically acceptable. The term “pharmaceutically acceptable”, as used herein, refers to the non-toxicity of a material, which, preferably, does not interact with the action of the active component of the pharmaceutical composition, i.e., the AAV vector or host cell of the present invention. In particular, “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia, European Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to an organic or inorganic component, of a natural or synthetic nature, in which the active component is combined in order to facilitate, enhance or enable application. According to the invention, the term “carrier” also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a subject. Possible carrier substances (e.g., diluents) are, for example, sterile water, Ringer's solution, Lactated Ringer's solution, physiological saline, bacteriostatic saline (e.g., saline containing 0.9% benzyl alcohol), phosphate-buffered saline (PBS), Hank's solution, fixed oils, polyalkylene glycols, hydrogenated naphthalenes and biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers. In one embodiment, the carrier is PBS. The resulting solutions or suspensions are preferably isotonic to the blood of the recipient. Suitable carriers and their formulations are described in greater detail in Remington's Pharmaceutical Sciences, 17^(th) ed., 1985, Mack Publishing Co. The term “excipient”, as used herein, is intended to include all substances which may be present in a pharmaceutical composition and which are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffer substances, stabilizing agents, flavouring agents or colorants.

The present invention further provides a kit comprising the AAV vector or a nucleic acid molecule, e.g., the AAV vector construct, or the host cell or the pharmaceutical composition of the present invention. As used herein, the term “kit” (also referred to as “kit of parts”) refers to an article of manufacture comprising one or more containers and, optionally, a data carrier. Said one or more containers may be filled with one or more of the means or reagents disclosed herein, e.g. one container with an AAV vector of the present invention and one container with the corresponding activator molecule. Additional containers may be included in the kit that contain, e.g., diluents, buffers and further reagents. Said data carrier may be a non-electronical data carrier, e.g., a graphical data carrier such as an information leaflet, an information sheet, a bar code or an access code, or an electronical data carrier such as a floppy disk, a compact disk (CD), a digital versatile disk (DVD), a microchip or another semiconductor-based electronical data carrier. The access code may allow the access to a database, e.g., an internet database, a centralized, or a decentralized database. Said data carrier may comprise instructions for the use of the kit in accordance with the present invention.

The inventor has surprisingly found that the AAV vectors of the present invention allow for the regulatable expression of a molecule of interest, e.g., a therapeutically active peptide or protein or a therapeutically active oligo- or polynucleotide, with very low or even zero expression in the non-induced state (“zero background expression”). Such AAV vectors will allow gene therapeutic approaches that are more safe and have less unwanted side effects than current AAV vector-based approaches.

The present invention is further illustrated by the following examples, which are not to be construed as limiting the scope of the invention.

Examples

Several different configurations of a one-vector GS-GDNF layout were designed, which were packaged into AAV-5 viral capsids and tested in the rat brain for GDNF production in absence or presence of Mfp. FIG. 2 depicts some of the constructs that were finally assessed, Table 1 shows GDNF levels obtained from rat brains before and after induction with Mfp.

TABLE 1 GFNF levels as determined by ELISA in rat brains, injected with AAV-5 viruses at specified titres. Mfp was given at 3 weeks after vector injection, tissues were prepared at 1 week after Mfp application. Vector injected into GDNF − Mfp (pg/mg GDNF + Mfp rat brain Titre (vg) tissue) (pg/mg tissue) no vector — 5 5 # 17 3 × 10e9 650 1100 # 20 3 × 10e9 175 900 # 21 3 × 10e9 9 1900 # 21 1 × 10e9 5 1300

The results shown in Table 1 demonstrate that the layout of vector #17 (see FIG. 2) made it almost useless for regulated gene therapy, as in the non-induced state it produced GDNF levels 130-fold over background, while Mfp induction only increased GDNF levels 2-fold further. Insertion of a small synthetic polynucleotide containing insulator sequences (TB=transcription pause and polyadenylation sites; see vector #20) significantly reduced non-induced GDNF levels and improved the rate of induction, but non-induced GDNF levels were still 35-fold higher than normal rat GDNF brain levels.

Vector #21 contains the same elements as vector #20, except that the expression cassette for GS is inverted, i.e., the two expression cassettes are arranged in a tail-to-head configuration (see FIG. 2 and Table 2). This feature provided dramatically improved performance of the vector, in that non-induced GDNF levels were only 2-fold increased as compared to normal background GDNF levels in the rat brain, while induced GDNF levels were 380-fold over background. Reducing the injected virus titre from 3×10e9 vector genomes to 1×10e9 vector genomes resulted in non-induced GDNF levels indistinguishable from normal rat brain levels, with still 260-fold induction. At four weeks after Mfp induction, GDNF levels in rats injected with AAV-5 #21 at 1×10e9 vg had returned to background levels again. To the inventor's knowledge, this is the first description of any regulated gene transfer system in AAV vectors with zero background expression in the non-induced state, and the first functional one-vector genome layout of the gene switch system in AAV vectors.

The vector genome of the invention (see, for example, SEQ ID NOs: 1 and 2) contains unique restriction sites at strategic positions, allowing easy further manipulation of its layout, for example taking away splice sites for reduced expression levels of the GS fusion protein as well as exchange of promoter elements and/or the transgene/molecule of interest.

TABLE 2 Annotated sequence of an exemplary vector genome of the present invention (vector #21; SEQ ID NO: 1). In the recombinant virus, i.e., in the complete AAV vector, this sequence was flanked by the AAV-2 inverted terminal repeats. BglII         MluI     BamHI       HindIII          PstI    ApaI ------       ------    ------       ------         ------- ------    1 agatctagga tcacgcgtaa aggatccaaa aaaaagctta aactagactg cagagggccc tgcgtatgag                                               >>......′HSYN-Promoter.......>   71 tgcaagtggg ttttaggacc aggatgaggc ggggtggggg tgcctacctg acgaccgacc ccgacccact >..............................′HSYN-Promoter..............................>  141 ggacaagcac ccaaccccca ttccccaaat tgcgcatccc ctatcagaga gggggagggg aaacaggatg >..............................′HSYN-Promoter..............................>  211 cggcgaggcg cgtgcgcact gccagcttca gcaccgcgga cagtgccttc gcccccgcct ggcggcgcgc >..............................′HSYN-Promoter..............................>  281 gccaccgccg cctcagcact gaaggcgcgc tgacgtcact cgccggtccc ccgcaaactc cccttcccgg >..............................′HSYN-Promoter..............................>  351 ccaccttggt cgcgtccgcg ccgccgccgg cccagccgga ccgcaccacg cgaggcgcga gatagggggg >..............................′HSYN-Promoter..............................>  421 cacgggcgcg accatctgcg ctgcggcgcc ggcgactcag cgctgcctca gtctgcggtg ggcagcggag >..............................′HSYN-Promoter..............................>                                        NheI           AgeI         NcoI                                       -------        -------      ------              491 gagtcgtgtc gtgcctgaga gcgcagtcga aagctgctag caaccatcca ccggtcgcca ccatggatag >........′HSYN-Promoter.........>>                                                                     >>.....>  561 ccagcagccc gatctgaaat tgctgtcctc tattgaacag gcttgtgata tttgcaggct taaaaaactc >.................................pSwitch..................................>  631 aaatgttcca aagaaaaacc aaagtgtgct aaatgtctga agaacaactg ggaatgccgc tactccccca >.................................pSwitch..................................>  701 agaccaagcg ttctccactt actcgcgctc acctgacaga ggtagaaagt aggctggaac gcctagaaca >.................................pSwitch..................................>                                                                       EcoRV                                                                       ------  771 gttgtttttg ttgatattcc caagagaaga cctggacatg attcttaaga tggatagcct gcaagatatc >.................................pSwitch..................................>  841 aaggcgctcc tggagtttcc aggcgtcgat cagaaaaagt ttaacaaggt ccgagtcgtc cgagccctag >.................................pSwitch..................................>  911 acgccgttgc cctccctcaa cccgtgggcg tgcctaatga aagccaggct ctctcacagc ggtttacttt >.................................pSwitch..................................>  981 cagcccaggg caggatatac agcttatacc tcccctgata aatttattga tgagtatcga gccggacgtg >.................................pSwitch..................................> 1051 atttacgcag ggcatgataa cactaagcca gacacatctt cttctctcct gaccagccta aaccaactgg >.................................pSwitch..................................> 1121 gtgaacggca gcttctgtca gtcgtgaagt ggagcaaatc cctccctgga tttagaaacc tgcacataga >.................................pSwitch..................................> 1191 tgaccaaata acacttattc aatactcctg gatgagctta atggtgtttg gtctcggatg gcggtcatat >.................................pSwitch..................................> 1261 aagcacgtta gcggccagat gctctacttt gcccctgatt tgattctgaa cgaacaaagg atgaaggaga >.................................pSwitch..................................> 1331 gctccttcta tagcctttgt ctgacgatgt ggcaaatccc gcaggagttt gtaaaactgc aagtgagtca >.................................pSwitch..................................> 1401 ggaggagttc ctgtgtatga aagttctact gctgctcaat acgatcccct tggaagggct cagatcacag >.................................pSwitch..................................> 1471 acgcaattcg aagagatgag gagctcttat attagagagc taattaaggc tattggtctg aggcaaaagg >.................................pSwitch..................................> 1541 gtgtcgtgtc cagcagccag agattttacc aacttactaa actactggac aacctacacg acctagtcaa >.................................pSwitch..................................> 1611 gcagctccat ctctactgcc tgaacacctt cattcaatcc agagccttat ctgtggaatt tccggaaatg >.................................pSwitch..................................>                                 NcoI                               ------- 1681 atgagtgagg tcattgcggg gtcaactccc atggaatttc agtatctgcc agacaccgat gacaggcacc >.................................pSwitch..................................> 1751 gcatcgaaga gaaacggaaa cggacatacg agaccttcaa gtccattatg aaaaagagtc ccttttctgg >.................................pSwitch..................................> 1821 ccctaccgac cccagacccc ctccaaggag aatcgcagtg ccttccagga gtagtgcatc agttccgaaa >.................................pSwitch..................................>                                                                       NcoI                                                                      ------- 1891 ccggccccac agccatatcc ttttaccagt tctctgagta ccattaacta cgacgaattt cctaccatgg >.................................pSwitch..................................> 1961 ttttcccctc ggggcaaatt agccaggcgt ccgcgctggc gcccgcgccc ccccaggtcc ttccgcaggc >.................................pSwitch..................................> 2031 tccagcccct gctccagccc ccgcaatggt ttctgccctg gcacaagccc ccgcacccgt gcctgtgttg >.................................pSwitch..................................> 2101 gcaccaggcc cgccacaggc cgtggcacct ccggctccta aacctactca ggccggagag ggcaccctta >.................................pSwitch..................................> 2171 gtgaggccct gttacagctt caatttgacg acgaggacct cggggctctc cttggcaatt cgacagaccc >.................................pSwitch..................................> 2241 cgctgtgttt acagacctgg cttcggtaga caattctgag tttcagcaac ttctcaacca gggcatcccc >.................................pSwitch..................................>                                                                  BstEII                                                                 -------- 2311 gtagcccctc atacaacaga gcccatgtta atggagtacc cagaggctat cacaaggctg gtaaccggcg >.................................pSwitch..................................> 2381 cccaaagacc accagatcct gcaccagcac cactgggagc tcctggttta cccaatggat tattatcagg >.................................pSwitch..................................>                                                                           SpeI                                                                          ------ 2451 agatgaggat ttcagttcca ttgccgatat ggacttcagc gcgcttcttt ctcagatcag ctcttgaact >...............................pSwitch..................................>           BlnI          -------        StuI       ------- 2521 agtaaaaggc ctaggtaagt atcaaggtta caagacaggt ttaaggagac cacatagaaa ctgggcttgt       <...............................′INTRON..............................>                >> 2591 cgagacagag aagactcttg cgtttctgat aggcacctat tggtcttact gacatccact ttgcctttct >.................................′INTRON..................................>                                  >>               StuI                             PsiI              ------                           ------ 2661 ctccacaggt gtaggccttt cgagcaactt gtttattgca gcttataatg gttacaaata aagcaatagc >.′INTRON.>>       >>...>>              >>..........................SV40-pA.>>>>>>>>>>>>>>>>>>>>>>>>>>> 2731 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg >.................................SV40-pA..................................>                                                >>                                                           XbaI                                                          ------ 2801 tatcttatca tgtctggatc gtctagcatc gaagatccac tagatgcata aatctagaca ataaaatatc >.............SV40-pA.............>>                                                                 >>...TB′...>                                            ClaI                                           ------ 2871 tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac taacatacgc tctccatcaa >..................TB′.....................>>                                              >>............′TB.............> 2941 aacaaaacga aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag tgcaggtgcc agaacatttc >...................................′TB....................................>         ApaI        ------- 3011 tctattaggg cccaagcgga gtactgtcct ccgagtggag tactgtcctc cgagcggagt actgtcctcc >>>                  >>.....GAL4....>>                                       >>.....GAL4 ....>>                                                            >>.....GAL4.....> 3081 gagtcgaggg tcgaagcgga gtactgtcct ccgagtggag tactgtcctc cgagcggagt actgtcctcc >                  >>.....GAL4....>>                                       >>.....GAL4 ....>>                                                            >>.....GAL4.....>                                    EcoRV                                    ......                               XhoI                              ------- 3151 gagtcgacta gagggtatat aatggatctc gagatatcgg agctcgttta gtgaaccgtc agatcgcctg >            >>...TATA....>>                                             <<.............................>                                                                > +1 of 1.91E transcript 3221 gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg cggccgggaa >....................HCMV IE 3 from TATA (mRNA 5end).....................>                                PacI                               --------- 3291 cggtgcattg gaacgcgcat tccccgtgtt aattaacagg taagtgtctt cctcctgttt ccttcccctg >...........................>>                                           >>IVS8′..........................>                                PstI                 NheI                               -------              ------- 3361 ctattctgct caaccttcct atcagaaact gcagtatctg tatttttgct agcagtaata ctaacggttc >..................................IVS8′....................................>                               KpnI                              -------                          MfeI             AgeI        NcoI                         ------           -------     ------- 3431 tttttttctc ttcacaggcc accaattggt accgagctac cggtcgccac catgggaaag ttatgggatg >.....IVS8′.....>>                                                         >>......GDNF.......> 3501 tcgtggctgt ctgcctggtg ttgctccaca ccgcgtctgc cttcccgctg cccgccggta agaggcttct >.................................GDNF.....................................> 3571 cgaagcgccc gccgaagacc actccctcgg ccaccgccgc gtgcccttcg cgctgaccag tgactccaat >.................................GDNF.....................................> 3641 atgcccgaag attatcctga ccagtttgat gacgtcatgg attttattca agccaccatc aaaagactga >.................................GDNF.....................................>    BstEII                                                      PstI    -------                                                    ------- 3711 aaaggtcacc agataaacaa gcggcggcac ttcctcgaag agagaggaac cggcaagctg cagctgccag >.................................GDNF.....................................> 3781 cccagagaac agcagaggga aaggtcgcag aggccagagg ggcaaaaatc gggggtgcgt cttaactgca >.................................GDNF.....................................> 3851 atacacttaa atgtcactga cttgggtttg ggctacgaaa ccaaggagga actgatcttt cgatattgta >.................................GDNF.....................................> 3921 gcggttcctg tgaagcggcc gagacaatgt acgacaaaat actaaaaaat ctgtctcgaa gtagaaggct >.................................GDNF.....................................> 3991 aacaagtgac aaggtaggcc aggcatgttg caggccggtc gccttcgacg acgacctgtc gtttttagac >.................................GDNF.....................................>                                                                     NotI                                                                   -------- 4061 gacagcctgg tttaccatat cctaagaaag cattccgcta aacggtgtgg atgtatctga gcggccgcac >-----------------------------GDNF.............................>>                    BclI                   ------- 4131 cgtcgactag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc                 >>.........................bGH pA..........................> 4201 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca >..................................bGH pA..................................> 4271 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt >..................................bGH pA..................................>                     SphI         BglII                   -------        ------ 4341 gggaagacaa tagcagggca tgctggggag agatct >....bGH pA....>> 

1. An adeno-associated virus (AAV) vector comprising (i) a first expression cassette directing the expression of a regulator protein under the control of a first promoter, wherein the regulator protein is activated in the presence of an activator molecule, and (ii) a second expression cassette directing the expression of a molecule of interest, wherein the second expression cassette comprises a promoter region, and the expression of the molecule of interest is induced by binding of the activated regulator protein to the promoter region, wherein the first expression cassette and the second expression cassette are arranged in a tail-to-head configuration.
 2. The AAV vector of claim 1, wherein the first promoter has one or more of the following features: (i) it is a constitutive promoter; (ii) it is selected from the group consisting of cell-specific promoters, tissue-specific promoters and organ-specific promoters; (iii) it is selected from the group consisting of human synapsin 1 gene (hSYN1) promoter, tubulin alpha 1 (Ta1) promoter, glial fibrillary acidic protein (GFAP) promoter, cytomegalovirus (CMV) promoter, human beta-actin-CMV hybrid promoter and functional fragments or variants of any of the foregoing.
 3. The AAV vector of claim 1, wherein the regulator protein is a gene switch fusion protein comprising a GAL4 DNA binding domain, a truncated progesterone receptor ligand binding domain and a p65 transactivation domain from NF-kappaB.
 4. The AAV vector of claim 3, wherein the activator molecule is mifepristone (Mfp).
 5. The AAV vector of claim 1, wherein the first expression cassette comprises, in 5′ to 3′ direction, the first promoter, a coding sequence for the regulator protein and a first polyadenylation signal sequence.
 6. The AAV vector of claim 5, wherein the first expression cassette further comprises a synthetic intron arranged between the coding sequence for the regulator protein and the first polyadenylation signal sequence.
 7. The AAV vector of claim 1, wherein the promoter region comprises one or more binding sites for the activated regulator protein and a second promoter.
 8. The AAV vector of claim 7, wherein the promoter region further comprises a synthetic intron.
 9. The AAV vector of claim 7, wherein the one or more binding sites for the activated regulator protein are GAL4 binding sites.
 10. The AAV vector of claim 7, wherein the second promoter is a minimal promoter which is induced by the binding of the activated regulator protein to the one or more binding sites for the activated regulator protein.
 11. The AAV vector of claim 10, wherein the second promoter is a minimal promoter comprising a TATA sequence and/or an mRNA initiation sequence.
 12. The AAV vector of claim 1, wherein the molecule of interest is a therapeutically active peptide or protein or a therapeutically active oligo- or polynucleotide.
 13. The AAV vector of claim 12, wherein the molecule of interest is a neurotrophic factor.
 14. The AAV vector of claim 13, wherein the neurotrophic factor is glial cell line-derived neurotrophic factor (GDNF).
 15. The AAV vector of claim 1, wherein the second expression cassette comprises, in 5′ to 3′ direction, the promoter region, a coding sequence for the molecule of interest and a second polyadenylation signal sequence.
 16. The AAV vector of claim 1, wherein the first expression cassette and the second expression cassette are separated by a nucleotide sequence comprising an insulator element.
 17. The AAV vector of claim 16, wherein the insulator element is a transcription blocker comprising a transcription pause site and a polyadenylation signal sequence.
 18. The AAV vector of claim 1, wherein the AAV vector comprises the nucleotide sequence represented by SEQ ID NO: 1 or a functional variant thereof, wherein the functional variant has a nucleotide sequence which is at least 80% or at least 85% or at least 90% or at least 95% identical to SEQ ID NO:
 1. 19. The AAV vector of claim 1, wherein, in the absence of the activator molecule, the molecule of interest is not expressed in a host or is expressed in a host at a level which is at most 10-fold or at most 5-fold or at most 4-fold or at most 3-fold or at most 2-fold increased as compared to the normal expression level of the molecule of interest in the host, wherein the host is a cell, tissue or organ.
 20. An adeno-associated virus (AAV) vector construct comprising (i) a first expression cassette directing the expression of a regulator protein under the control of a first promoter, wherein the regulator protein is activated in the presence of an activator molecule, and (ii) a second expression cassette comprising a multiple cloning site allowing the insertion of a coding sequence for a molecule of interest, wherein the second expression cassette comprises a promoter region, and the expression of the molecule of interest is induced by binding of the activated regulator protein to the promoter region, wherein the first expression cassette and the second expression cassette are arranged in a tail-to-head configuration.
 21. The AAV vector construct of claim 12, wherein the AAV vector construct comprises the nucleotide sequence represented by SEQ ID NO: 2 or a functional variant thereof, wherein the functional variant has a nucleotide sequence which is at least 80% or at least 85% or at least 90% or at least 95% identical to SEQ ID NO:
 2. 22. A method for treating, ameliorating or preventing a disease or disorder in a subject, comprising (a) introducing into the subject the AAV vector of claim 1; and (b) administering to the subject the activator molecule to induce expression of the molecule of interest, wherein the molecule of interest is a therapeutically active peptide or protein or a therapeutically active oligo- or polynucleotide.
 23. The method of claim 22, wherein the molecule of interest is a neurotrophic factor and the disease or disorder is selected from the group consisting of Parkinson's disease, Huntington's disease, spinal cord lesion and an amyloid-related disorder.
 24. The method of claim 23, wherein the neurotrophic factor is glial cell line-derived neurotrophic factor (GDNF).
 25. The method of claim 23, wherein the amyloid-related disorder is selected from the group consisting of Alzheimer's disease, cerebral amyloid angiopathy, dementia, motor neuropathy, Down's syndrome, Creutzfeld Jacob disease, transmissible spongiform encephalopathies, hereditary cerebral hemorrhage with amyloidosis Dutch type, HIV-related dementia, fronto-temporal dementia, Lewy body disease, mixed dementias, head trauma, familial Danish Dementia, familial British Dementia, inclusion body myositis (IBM), neuronal disorder related to aging, and chronic pain. 